how to remove affinity tag completely - (Oct/16/2013 )
Hello everyone,
I am new here and very glad to be a member here.
There is a frustrating problem with my project. I used His tag and MBP tag to get my target protein.Schematic diagram as:
---HIS---MBP---HRV 3C cleavage site--my target protein---
After purification, I use HRV 3C to cut away the tags, then load onto Ni-NTA column to remove the tags and collect the flow through.
The ideal thing is all of the tags will be attached to the column and my pure protein is in flow through, but actually there is always a small part of tags can not bind to the beads. I do not know what happens. It is very confusing because the protein with these tags is just get from the Ni-NTA, and now the tags can not bind to it! Although I tried that there is no imidazole in the sample, the tags can not bind, either. If anyone can help me and give me some suggestions.
Thank you very much.
Can you do an immunoprecipitation for your tag? Should leave all untagged protein in your lysate.
Did you try loading your protein on a nickel column again. Possibly you have overloaded your column, or the equilibrium is such that some sneaks through.
You could increase the binding by making the his region longer than the typical six AA, or by adding a second region.
jerryshelly1 on Wed Oct 16 18:39:22 2013 said:
Can you do an immunoprecipitation for your tag? Should leave all untagged protein in your lysate.
Thank you for your reply. I should make sure that the purity of target protein is high because 1) the protein is used for electron microscope and the molecular weight is twice smaller than the tag. 2) if possible, I want to do crystal screen with my protein. So I don't know if the antibody can be a new kind of contaminate and I should know the exactly number of the antibody.
phage434 on Wed Oct 16 23:12:04 2013 said:
Did you try loading your protein on a nickel column again. Possibly you have overloaded your column, or the equilibrium is such that some sneaks through.
You could increase the binding by making the his region longer than the typical six AA, or by adding a second region.
I am appreciated that you reply this. I really tried another loading, but it didn't work. And I think the volumn of my Ni-NTA beads was enough. Also, I made the construct with 8 HIS and 10 HIS. Either of them worked. My protein has a tendency to form cluster. So maybe tags can not be removed are buried by my protein, I think.
You could bind your protein to a maltose column, with the MBP domain. Then, you could wash extensively and then cut with your protease, and elute the desired protein off the column. This might work better with a different binding domain. The chitin binding domain used in the NEB IMPACT kit works well, but MBP might work just fine.
phage434 on Thu Oct 17 14:53:27 2013 said:
You could bind your protein to a maltose column, with the MBP domain. Then, you could wash extensively and then cut with your protease, and elute the desired protein off the column. This might work better with a different binding domain. The chitin binding domain used in the NEB IMPACT kit works well, but MBP might work just fine.
Thanks a lot. Actually I did do the on-column cleavage with MBP beads. It didn't work,either. Some part of tags can not bind.......I don't know about the NEB IMPACT kit, I will check it out. Thank you again for your patience. Wish you good time.
If some part of your tagged protein sample fails to bind to the maltose column, then you should be able to wash it away after binding to the column. You may need to do this with reduced salt to minimize protein-protein binding. I don't understand how uncut protein escapes after this wash. In any case, following this wash, you should be able to pull out any protein with the his tag using nickel beads. Maybe I'm missing something.
Dear Hellen,
Can i know the estimated protein size of your targeted protein without tag?
I am a little curious, had you try this before?
1) Load recombinant protein into the Ni-NTA or MBP column
2) Wash the unbound flow through
3) Add in your HRV3c (around < 2 CV) into your column. Hold the system for 30 minutes (or any amount of time desired for the protease to do it's work)
4) Instead of using elution buffer, perform an isocratic elution (for around 5 CV) using column / running buffer and start collecting all the fractions
5) Pull all your fractions together, perform size exclusion chromatography (Superdex Peptide PC 3.2/30 -or- HiLoad 16/600 Superdex 75 pg) to separate your HRV3C and your protein of interest.
CV : Column Volume
Adrian K on Sat Oct 19 17:17:20 2013 said:
Dear Hellen,
Can i know the estimated protein size of your targeted protein without tag?
I am a little curious, had you try this before?
1) Load recombinant protein into the Ni-NTA or MBP column
2) Wash the unbound flow through
3) Add in your HRV3c (around < 2 CV) into your column. Hold the system for 30 minutes (or any amount of time desired for the protease to do it's work)
4) Instead of using elution buffer, perform an isocratic elution (for around 5 CV) using column / running buffer and start collecting all the fractions
5) Pull all your fractions together, perform size exclusion chromatography (Superdex Peptide PC 3.2/30 -or- HiLoad 16/600 Superdex 75 pg) to separate your HRV3C and your protein of interest.
CV : Column Volume
Thank you very much. My target protein without tag is about 28kD.
1) I used Ni-NTA and MBP column to get my quite pure protein with tag. Then used HRV3C(it has his8 tag) to cut the tag. After this, I load the mixture onto Ni-NTA again. Theoretically as long as the NI-NTA medium is enough, the uncut protein, the tag and the HIS-HRV3C will be attached, and my protein flow through. But there was a part of tag also flowed through. So I planned to do the on-column cleavage use these two kinds of column. 2)After loading my protein lysates onto the columns and washing the unbound contaminant, I added HRV3C into the columns and hold them for about 12h. For Ni column, nothing flowed through. Maybe because the number of His. But for MBP column, a small part of tag also flowed through although most can bind. 3)Also, I tried resource Q column to separate the tag and my protein. The result was confusing. Although I got two peaks, each peak contains the tag and my protein,and it seems to be the same ratio. Maybe I didn't get the appropriate condition for this.4)I also tried gel filtration(Superdex 75 or Superdex 200). All of the proteins are in the high aggregation peak, include the MBP tag.......I am sorry about that I do not know how to attach the result image.... I am very frustrated.
Thank you again for your warm-hearted reply.